The introduction of new improved electronic counting devices has shown the need for improvement in quality control products, especially for human platelet reference controls for enumeration of cellular biological components for clinical diagnostic purposes. One such system is that of the Coulter.RTM. S-Plus hematology system that counts and examines the volume distribution of human platelets in whole blood specimens. Instruments of similar type are sold by other manufacturers for this purpose.
Early examinations, utilizing the Coulter.RTM. Channelyzer.RTM. system for volume distribution analysis, revealed variations in size distributions of platelets from different human platelet rich plasma preparations. Shifts in the mode of the platelet distribution curves demonstrated count discrepancy. These observations raised questions as to their significance in clinical application.
Mean cell volume distribution analysis has gained recognition recently as a useful measurement for clinical application. Human platelet distribution width can now be calculated from specifically measured parameters. Each of these measured parameters lends itself to improvement in examining the log normal distribution of human platelets.
Specifications as to electronic calibration are well defined to the operator of such instruments. However, reference controls are needed to assure good quality control procedures and to monitor functional characteristics with respect to count modes and volume distribution analysis.
It is extremely important to adequately monitor these hematology systems. Any human platelet reference control must satisfy all the criteria that are measured on human patient specimens. The reference control must simulate as closely as possible that of normal fresh whole blood specimens. It must also conform to properly set thresholds as to the count mode, correctly balanced apertures, current, amplification gain settings, and responsiveness of the system as to all functional aspects.
Several reference control preparations are currently marketed; however, they lack long term stability as to count, mean cell volume, and size distribution. Also, changes and shifts in the mode result in erronneous counts. The common cause of count discrepancies is aggregation. Aggregation creates doublets that count in the white blood cell mode resulting in unreliable quality control measures. Still another discrepancy in count occurs due to poor performance of stability with time until the expected expiration date.
Attempts to stabilize human platelets have proved to be extremely difficult. One major problem is disintegration of the platelet membrane. When platelet membranes disintegrate, they cause debris which result in erroneous counts. New methods through improved computer technology for curve fitting of raw data are beset by these problems.
In U.S. Pat. No. 4,160,644 (1979), Ryan to Streck Laboratories, Inc., a method of preparing a reference control composition is described which is stable as to count and agitation in which a minor amount of solid polyethylene glycol, is added, either to a platelet suspension which has been fixed with glutaraldehyde, or to the diluent for this aldehyde treated platelet suspension, in order to achieve stabilization due to time and also during agitation. The preferred solid polyethylene glycol has a molecular weight of about 6000. It was reported that the lower molecular weight, liquid polyethylene glycols were ineffective for this purpose, even though the effect on the surface tension was similar. It was further reported in this patent that "an examination of the size of the surfactant treated platelets in a Coulter.RTM. ZBI, an instrument which is used for size analysis of particles such as platelets and white blood cells, indicated that there was a large decrease in the size of the platelet. However, when platelets were examined under the microscope with an ocular-micrometer, no change in size of the platelet could be determined. The principle of the Coulter Counter.RTM. is that it measures changes in conductivity; the surfactants alter the conductivity and thus make the platelet appear smaller to the instrument." The inventors of the present invention do not agree with this finding. They report that change in actual volume (due to shrinking) would not be distinguishable by use of a microscope which looks at diameter, because in a platelet-sized particle, a small change in diameter results in a large change in volume. They believe that the actual effect of surfactants is to cause a shape change, e.g., transition from discoidal to spherical, which changes apparent volume by a factor of 1:4.
In U.S. Pat. No. 4,198,206 to Ryan (1980), the aldehyde treated suspension is washed with a solution of
(1) an amino acid which is glycine or alanine PA1 (2) glycol, glycerol or methanol PA1 (3) buffer salts PA1 (4) a solid polyethylene glycol (molecular weight 4,000 to 20,000)
in order to produce platelets that do not aggregate, and that maintain their size for at least 6 months.
An aqueous solution of glutaraldehyde and non-ionic surfactant which is a mixture of ethoxylates of isomeric linear alcohols is described in U.S. Pat. No. 3,912,450 (1975) and U.S. Pat. No. 3,968,248 (1976), Boucher to Wave Energy Systems, Inc. These patents make no mention of the use of such a composition for hematological purposes, but only for sterilization.
A large family of analytes has been employed by other investigators to increase the rigidity by fixation of cellular membranes. Examples of these fixative agents are formaldehyde, glutaraldehyde, pyruvic aldehyde, and similar compounds. Use of such components may alter the size distribution and volume of human platelets. Aggregation of the platelets is an ever present problem, in addition to long term stability of the fixed platelets, limiting the usefulness of such compounds in improved quality control measures.
The present invention relates to a method of stabilizing human platelets which lends itself to eliminate these aforementioned discrepancies, so that the reporting of patient results to the diagnostic clinician can be assured of correct results, resulting in improved health care and improvements in the health care industry.